2, 2&#39;, 3, 3&#39;, 4, 4&#39;, 6, 6&#39;-octahydroxy-5, 5&#39;-dicarboxy diphenylmethane



Patented Apr. 13, 1954 2,2',3,3,4,4,6,6'-OCTAHYDROXY- 5,5'-DICAR- BOXY DIPHENYLMETHAN E Ladislaus Arthur Hahn, Malmo, Sweden, and

' Janos Fekete, Lorena,

Brazil, assignors to Aktiebolaget Ferrosan, Malmo, Sweden, a corporation of Sweden N Drawing. Application September 9, 1952, Serial No. 308,703

Claims priority, application Sweden December 9, 1949 1 Claim. 1

This application is a continuation-in-part of our co-pending application No. 199,564, filed December 6, 1950.

The present invention relates to new biologically active diphenylmethane derivatives. and to a method of making the same.

An enzyme which is capable of splitting mucopolysaccharides and amongst them particularly the hyaluronic acid has been discovered i. a. in the testicles and the sperm of mammals, furthermore in the leech, in serpents and insects poisons and in certain bacteria. The enzyme has been denominated mucinase but the name has later been changed to hyaluronidase, or mucopolysaccharase, respectively.

The substrate hyaluronic acid is rather com mon in the animal organism. Among the most important sources may be mentioned the interfibrillar substance of mesenchymal tissues, especially of the connective tissues, furthermore the vitreous body of the eye, the synovia and the gel that surrounds the ovum on its way through the oviduct. Another important substrate for the hyaluronidase, the chondroitin sulfuric acid, is a substantial component of all sorts of cartilage, thus also of the cartilage of the joints.

The splitting effect of the hyaluronidase on the hyaluronic acid has been elucidated to a large extent: the highly polymeric acid is depolymerized and split into smaller units, the glucosidic bonds between the elements of the hyaluronic acid, i. e. the glucuronic and the N-acetyl glucoseamine, being hydrolyzed. In the complete splitting up of the hyaluronic acid to monosaccharide units also other enzymes are active. One of these enzymes has been isolated from bulls testicles and has been denominated mucooligosaccharase. The splitting of the hyaluronic acid and of other mucopolysaccharides, for instance the chondroitin sulfuric acid, through hyaluronidase may be experimentally shown and quantitatively determined for instance (a) by observing the decrease in viscosity of the substrate or (b) by determining the reducing groups which are liberated at the hydrolysis. There is also a biological method of determination which is based upon the influence of the hyaluronidase on the permeability of the skin, said influence being explained in the following.

The effect of the hyaluronidase in vivo corresponds to its effect on mucopolysaccharides in vitroff: for instance the hyaluronic acid of the skin is split by the action of hyaluronidase, which is brought into the tissue in one way or other, and looses its high viscosity. This causes the 56 skin to become much more permeable. This increase of the permeability is rather general and results therein, that as Well physiological as nonphysiological substances, such as water, salts, added chemical compounds of all kinds, coloring matters, poisons, bacteria and virus will spread many times quicker in the tissue if hyaluronidase is present. This may easily be shown experimentally by intracutaneous injection of 0.2 millilitres of Indian ink for instance on one side of the back of a rabbit, the same amount of ink to which there has been added some micrograms of hyaluronidase being injected on the other side. The ink to which no hyaluronidase has been added spreads only very slowly in the skin. The ink which has been mixed with the enzyme spreads in a few minutes over an area of 200 cm. or more. That which has been said about the effect of the hyaluronidase upon the permeability of the skin holds true also in respect of other kinds of mesenchymal tissue.

The high viscosity of the synovial fluid depends to a great extent on the hyaluronic acid and the same holds true i. a. of the vitreous body of the eye and of the gel that surrounds the ovum in the oviduct. The effect of the hyaluronidase upon such materials is to make them lose their viscosity so that they become fluent.

The hyaluronidase of certain bacteria is of great importance for various reasons. Some of the pathogenic bacteria containing hyaluronidase, such as streptococci, are connected with certain types of rheumatic diseases. Since the viscosity of the synovial fluid depends mainly on its content of hyaluronic acid and since the cartilage of the joints contains chondroitin sulfuric acid it was assumed that at least some forms of rheumatic and other joint diseases are caused directly or indirectly by hyaluronidase.

The effect of the hyaluronidase upon connective tissues results therein that bacteria containing or producing this enzyme will. penetrate through the organism much easier than other bacteria so that the risk of infection is highly increased. The hyaluronidase-bearing bacteria need not necessarily be pathogenic themselves, or they may be pathogenic only to a small degreenevertheless they may mean a great danger to the organism by facilitating the penetration of other non-physiological substances, such as other bacteria, virus etc., into tissues and organs. This is of particular importance since many highly pathogenic virus show a rather low penetration capability. If the organism is infected by hyaluronidas-producing bacteria infection with the said agents may result.

Since hyaluronic acid is present also in capillary walls the capillary permeability is influenced by hyaluron'idase. The hyaluronidase may therefore promote or facilitate the penetration of infective substances through the capillaries.

Certain substances inhibiting the effect of the hyaluronidase are known. As an example carboXy-p-benzoquinone, rutin, ascorbic acid, hepparin, hexylresorcinol, certain sera fractions and nitrates of hyaluronic acid may be mentioned.

It has now been discovered that an inhibition of the action of hyaluronidase which is many times stronger than that of the above-mentioned known substances is developed by certain diphenylmethane derivatives. These derivatives are obtained by condensing with formaldehyde 2,3,4,6-tetra-hydroxy benzoic acid. The inhibitory power against hyaluronidase of the product of this reaction is more than 30 times higher than that of any inhibitors earlier described. This very high inhibitory activity makes it possible to use such compounds inthe treatment. ,of rheumatoid arthritis on one side and infectious diseases induced byhyaluronidase producing bacteria on the other side. i

The inhibitor according to the invention may also be introduced into the organism in-a suitable manner, as per os, by superficial treatment or by subcutaneous, intramuscular or intraperitoneal injection etc. in order to prevent the spreading effect of the hyaluronidase in poisonings and infections. As mentioned above serpents and insects poisons. and various pathogenic bacteria contaimhyalur-onidase and in part also other mucopolysaccharases. If these enzymes get into the organism such as by biting or infection they hydrolyse the hyaluronic acid of the skin and other tissuesso thatv the poison or the bacteria are more easilyinfiltrated in the organism. Also infection with other infective substances which are free from hyaluronidase, such as bacteria and virus, spreads much quicker in the organism if. the same-is infected with hyaluronidase-bearing bacteria. lhe inhibitors according to this invention inhibit the actionof the hyaluronidase of poisons and bacteria upon tissues and thus prevent .or limit .infection.-. The action differs principally fromthe treatmentwith anti-toxins or anti-sera (no specificity)...

The compound which is the matter of the present invention is prepared in the following manner:

2.3.4.6-tetrahydroxy. benzoic acid is.,dissolved or suspended in a suitable solvent, .-for instance water, alcohol, acetic acid, hydrochloric acid of sulfuric acid. Then formaldehyde is added either all at one time or successivelyin the courseof the reaction. The presence of acondensation agent decreases the reaction time andgives bet.- ter yields. As a condensation agentmineral acids (for instance hydrochloric acid or sulfuric acid) or alkalis (for instance alkali hydroxides) may be used. The proportion of .thecondensation products here described. mayalsobecarried out in such a manner that'the carboxylic groups are introduced after the condensation.

Example 2 In grams of cold 50% sulfuric acid 18.6 grams of 2.3.4.6-tetrahydroxy benzoic acid are suspended. The suspension is placed on a boiling water bath and 4 grams of 40% formaldehyde are then added successively during 30 minutes. The reaction mixture is cooled. The precipitateiobtained washed with hot water and dried in vacuum. Yield: 11.0 grams of 2.2'.3.3;4.4'.6.6' octahydroxy-5;5'-dicarboxy diphenylmethane. Relative inhibitory power: 3650.

Example 3 In 100 grams of 10% by weight hydrochloric acid 14.2 grams of 1.2.3.5-benzene tetrol areflsuspended. 4 grams of 40% formaldehyde solution are added and the miXturewarmed-to. 60 'G. during an hour. tained is washed free from hydrochloric acid with cold water and dried. 10 grams of this-product which is methylenedi-benzenetetrol'ar'e mixed with 30 grams of potassium carbonate and heated in carbon dioxide atmosphere at a pressure of 4 atm. to 100C. during 4 hours. After cooling the reaction product is dissolved-in 'waterand precipitated by hydrochloric acid. After tworeprecipitations 2.223.3.44.6.6'-octahydroxy-5.'5- dicarboxy diphenylmethane is obtained infairly pure state. Yield: 4.1 grams.l Relative inhibitory power: 3700.

What we claim is: j-

A compound ofthe formula i o onjicoonr CE: 7 1' v I cubnpooofr A which is the condensation product of 2.3.4.6- tetrahydroxy benzoic .acid and formaldehyde. References Citedin the file 'of-this patent UNITED STATES PATENTS Number IIame Date.....:, 706,354 Summers Aug. 5,1902 2,042,343 Kyrides May zfi lgi fi OTHER REFERENCES Mohlau et al., Beilstein '(Handbuch, aches.) vol. 10, p.594 (1927):

The condensation product -ob-- 

